Molecular Mechanisms of Protein-Membrane Interactions Driving Insulin Secretion Project Summary/Abstract: This research project will determine the molecular mechanisms underlying protein-membrane interactions central to insulin secretion, including how these interactions are influenced by intracellular lipid changes associated with insulin secretory signaling. Membrane-targeting proteins are central to insulin secretion in the ? cells of the pancreatic islets of Langerhans, as well as signaling in many other cell types. One key membrane-targeting protein that plays a central role in secretion is synaptotagmin (Syt), which has two C2 domains that trigger secretory vesicle?plasma membrane fusion by docking to membranes in response to increased intracellular [Ca2+]. Most studies probing the molecular mechanisms of Syt activity have focused on Syt1, the major Syt isoform responsible for rapid neurotransmitter release. However, Syt1 does not play a major role in insulin secretion; rather, ? cells use Syt7, Syt9, and the Syt-like protein granuphilin for the insulin secretion pathway. The research proposed herein will test the hypothesis that the C2 domains from Syt7, Syt9, and granuphilin have biochemical and biophysical properties specialized for their roles in insulin secretion, and thus behave in a manner distinct from Syt1 despite having homologous structures. Prior results support this assertion, as the C2A domain from Syt7 binds membranes with a much stronger contribution from the hydrophobic effect than the corresponding domain from Syt1. The proposed studies will use a combination of established biochemical and biophysical techniques, along with cutting-edge single- molecule fluorescence microscopy, to probe the driving forces and molecular interactions underlying the activities of these C2 domains driving insulin secretion. In order to connect these mechanistic studies to cellular function and disease, the sensitivity of C2 domains to signaling lipids and oxidation products will also be investigated. A number of lipid signaling pathways are activated during glucose-stimulated insulin secretion, and the effects of major signaling lipids on Syt C2 domain membrane binding will be measured here. Proper control of C2 domain membrane interactions is vital for insulin secretion, and long-term alterations in these mechanisms could contribute to the loss of ? cell secretory function that accompanies type 2 diabetes. Overall, the results will both lead to a better understanding of insulin secretion pathways and shed light on possible mechanisms underlying ? cell defects in diabetes.